A major impediment in the molecular characterization of the Norwalk-like human caliciviruses (NLVs), viruses associated with epidemic gastroenteritis, has been the inability to identify a cell culture system that will support virus replication. A major goal of this project is to study molecular mechanisms of replication of the cultivatable feline calicivirus (FCV), that serves as an important model of calicivirus replication. This year, we continued characterization of the two minor proteins present in calicivirus virions, VPg and the protein encoded by ORF3. This work is relevant because it could lead to a better understanding of the initial stages of virus replication as well as RNA packaging and assembly of calicivirus particles. All caliciviruses, including the NLVs, possess a terminal ORF analogous to the FCV ORF3. The proteins encoded by these terminal ORFs show considerable variation in primary amino acid sequence and calculated molecular mass. However, they share an overall basic amino acid composition and were proposed to be involved in specific encapsidation of the caliciviral genome into particles through interactions with both capsid protein and the viral RNA. In our previous work, we had shown that serum raised to the protein encoded in the FCV ORF3 (calculated molecular mass is 12.2 kDa) recognized a 8.5 kDa protein in purified virions. Comparative analysis of radioactivity incorporated into virion proteins indicated the ORF3 protein is likely present in only one-two copies per virion. We demonstrated that the observed difference between the calculated and apparent masses in SDS-PAGE was not due to proteolytic processing of the protein and was likely due to an unusual mobility of the protein in SDS-PAGE. The mobility of the ORF3 protein present in virions was shown to be similar to that of the ORF3 protein found in FCV-infected cells or expressed in bacteria or in vitro. The function of the ORF3 protein is not known, but development of expression and purification protocols for this protein together with the availability of tools such as specific antisera and an infectious full-length cDNA clone will enable future studies of the functions of the ORF3 product. The second minor protein found in FCV virions, VPg, mapped to the region of ORF1 immediately upstream of the sequence encoding the viral proteinase. The protein identified in FCV virions had an apparent mass of 15.5 kDa. The smallest form of the VPg-related protein immunoprecipitated from FCV-infected cells was 14 kDa in SDS-PAGE and was apparently the predominant form of the VPg in FCV-infected cells. The presence of several forms of the VPg protein likely reflects modifications that the protein undergoes during viral replication. In addition, in infected cells, using an immunoprecipitation technique, we demonstrated the presence of VPg-containing precursor proteins with molecular masses of approximately 120, 90, 43, and 33 kDa. Further studies are in progress to establish the nature of the FCV VPg modification and its possible involvement in RNA replication.